The invention is related to an apparatus for conducting electrophysiological measurements on cells comprising a measuring head, the measuring head being provided with at least one electrode for impaling the cells.
An apparatus of the afore-mentioned type is disclosed in U.S. Pat. No. 6,048,722.
The prior art apparatus is used for conducting electrophysiological measurements on oocytes, in particular oocytes of Xenopus laevis, a South African claw frog. These oocytes are preferably used as expression systems for electrophysiological measurements. For that purpose the oocyte is placed and immobilized in a receptacle. The receptacle may, for example, be a funnel-type opening within a plate. Such plates are standardized and are conventionally used with 8xc3x9712=96, 16xc3x9724=384 or 32xc3x9748=1.536 receptacles (xe2x80x9cwellsxe2x80x9d). The receptacles for the oocytes may, for example, be provided with an opening at their bottom side, enabling to attract the oocyte by means of vacuum and to immobilize same within the receptacle.
For conducting electrophysiological measurements a genetic information, namely a mRNA or a cDNA is injected into the oocyte. As a consequence, characteristic ion channels and/or receptors are incorporated on the surface and within the oocyte which may be measured by applying an electrical voltage or directing an electrical current therethrough or by application of a substance.
It is well known to conduct pharmacological measurements in this manner because the receptors or ion channels developed in the membrane of the oocytes are configured in a kind that is characteristic for certain properties of the substances under investigation.
Within the apparatus according to U.S. Pat. No. 6,048,722 mentioned at the outset, a measuring electrode and a reference electrode are impaled into the cell. In addition a perfusion apparatus is connected to the receptacle of the cell. By means of this perfusion apparatus various substances, in particular measuring substances, may be filled into the receptacle in a controlled manner, the receptacle having typically a volume of 100 xcexcl.
Although this prior art apparatus is automatized for what concerns the supply of perfusion agents, it requires a considerable manual talent to bring the perfusion conduit into the area of the receptacle for the cell and to immobilize same at that location. Above all, the application of the electrodes to the cells, in particular impaling the electrodes into the cell, depends on the manual skill of the particular person conducting the experiment because these steps obviously have to be made manually. In case the application of the electrode or electrodes to the cell is unsuccessful, in particular, if the electrodes brake off, the entire experimental set up must be wholly re-assembled and re-adjusted. Finally, the prior art apparatus only allows to conduct individual measurements on one cell wherein only the sequence of different test fluids may be controlled automatically in a manner mentioned above.
It is, therefore, an object underlying the invention to improve an apparatus of the type specified at the outset such that the above-discussed disadvantages are avoided.
In particular it shall become possible to fully automatically conduct the experiments as well with respect to application of the electrodes as well as with respect to the application of the perfusion conduits. Further, the apparatus shall be adapted to be made ready for use with only a couple of manual operations according to the xe2x80x9cplug-and playxe2x80x9d principle and it shall be possible with the same principle that the apparatus can be put back to operation after a damage has happened.
Finally it shall become possible to execute a plurality of measurements on many different cells in a fully automatized mode, i.e. without supervision, in particular over night.
According to the apparatus specified at the outset this object is achieved by the present invention in that the electrodes are integrated into a common support.
This measure has the advantage that the carrier may be prefabricated on an industrial level and must, hence, only be plugged on a corresponding holder within the apparatus. The electrodes are already installed ready for operation, in particular with respect to their position relative to one another so that in contrast to conventional apparatuses there is no more need to delicately adjusting and orienting the electrodes relative to one another. By doing so the risk of damaging the electrodes during the setup of the apparatus is drastically minimized. Further, the measurements are clearly reproducible, because the electrodes are in a predetermined position relative to one another. Finally the integrated arrangement of the electrodes on the support allows that automatic displacement units for the measuring head thus obtained may be made, enabling to conduct the desired fully automatic measurements on a plurality of cells, for example in connection with a standardized multiwell-plate.
In preferred embodiments of the inventive apparatus the electrodes are inserted into recesses of the carrier, however, they may also be molded within the carrier.
This measure has the advantage that a stable and reproducible position of the electrodes within the carrier is achieved.
In other embodiments of the invention the electrodes consist of pulled glass tubes.
This measurement has the advantage that such glass electrodes may be configurated optimally for the specific application, as known per se. For example, it is possible to configurate the electrodes to have an electrical resistance of between 5 Mxcexa9 and 100 Mxcexa9, i.e. as so-called xe2x80x9csharp electrodesxe2x80x9d. As an alternative, the electrodes may be configurated as so-called xe2x80x9cpatch electrodesxe2x80x9d having an electrical resistance of the order of 500 kxcexa9 through 5 Mxcexa9.
As an alternative, the invention may provide to configurate the electrodes as wire electrodes, preferably as silver wire electrodes which, further preferably, are surrounded by a chloride coating. Besides that also tungsten wires or the like may be used.
In preferred embodiments of the inventive apparatus at least one electrode is provided with a straight section.
This measure has the advantage that the electrode may particularly easily be attached to the support.
In another preferred embodiment of the invention two electrodes are arranged essentially symmetrical relative to a longitudinal axis of the carrier so that the distance between the free ends of the electrodes are in a range of between about 50 xcexcm and 800 xcexcpm, preferably between 200 xcexcm and 500 xcexcm.
This measure has the advantage that during the introduction of the electrodes into the cells individual impaling holes are generated and not a common big hole which would be the case if the free ends of the electrodes would be arranged too close to one another. This would have the disadvantage that important physiological functions of the cells would go lost. With the inventive approach this is avoided.
In this connection it is particularly preferred when a straight section of the electrodes encloses an acute angle with a longitudinal axis of the support, the angle being in particular between 3xc2x0 and 10xc2x0, preferably 5xc2x0.
The inclined orientation of the electrodes with respect to one another has the advantage that the lower free ends of the electrodes may be positioned in a perfectly reproducible manner.
In a preferred group of embodiments of the invention at least one electrode is configured as a measuring electrode. The measuring electrode is preferably connected to a measuring amplifier which, further preferably, is adapted to be adjusted.
By doing so it is possible to conduct automatically con-trolled measurements of voltage and current signals.
This holds true in particular if the at least one measuring electrode is connected to a current source and the current source, in turn, is adapted to be adjusted.
This measure has the-advantage that if two different electrodes are used, the feeding of current on the one hand and the measuring of voltage on the other hand are decoupled one from the other.
The currents and voltages to be measured in the present context are in the nA through xcexcA range for currents and in the mV range for voltage measurements.
These measurements may be conducted by conventional electrophysiological measuring methods like xe2x80x9cbridge-modexe2x80x9d, xe2x80x9ccurrentclampxe2x80x9d, and xe2x80x9cvoltage-clampxe2x80x9d. In so far it is irrelevant for the invention whether the voltage clamp derivation is executed with two electrodes (two-electrode voltage clamp; TEVC) or with one electrode (single-electrode voltage-clamp, SEVC). In the SEVC method measurements are made in the so-called xe2x80x9cswitched-modexe2x80x9d, i.e. within a predetermined interval a measuring current is injected and the measuring voltage (with the current injection switched off) is alternately measured.
For that purpose it is preferred with further embodiments to at least configurate one of the electrodes as a reference electrode. In that case the reference electrode is preferably connected to ground.
In that case it is particularly preferred if at least two measuring electrodes are located in a first common plane and/or at least two reference electrodes are located in a second common plane and, further preferably, the first and the second plane extend parallel to each other and have a distance from each other which is as small as possible.
These measures have the advantage that an extremely compact design is achieved that is optimized from the point of view of the measuring technology and in which all necessary components are integrated as densely as possible.
In a further group of embodiments at least one perfusion conduit is arranged on the support.
This measure has the advantage that the perfusion apparatus is integrated into the same measuring head with respect to the feeding and discharging of the perfusion agent, the measuring head being the same in which the electrodes are already located so that all these elements may be handled altogether. Another advantage is that during the integration also of the perfusion conduits into the measuring head the relative positioning of the perfusion conduits relative to the electrodes may be optimized and may already be fixed in the manufacturing site. Also insofar a cumbersome handling of the apparatus during the setup of the measuring equipment has become obsolete. Further, one has the advantage that if one of the components should be damaged the entire measuring head may be replaced by another one with only a couple of manual operations.
According to variations of this group of embodiments the at least one perfusion conduit may be a perfusion inlet.
In that case it is particularly preferred when the perfusion inlet has a first end opening, when the perfusion inlet is arranged essentially parallel to the at least one electrode and if, finally, the first end opening is arranged above a lower end of the at least one measuring electrode.
This measure has the advantage that the perfusion agent is guided directly and exactly to the spot where the active portion of the measuring electrodes is located.
This holds true in particular if in connection with the already above-mentioned symmetrical arrangement of the electrodes the perfusion inlet is located essentially on the symmetry axis between the measuring electrodes.
It is, further, preferred in this context when the perfusion inlet is connected to a pump, the pump being preferably adapted to be adjusted.
This measure has the advantage that the perfusion agent may be supplied in precise quantities.
According to further variations of this embodiment the perfusion inlet is adapted to be connected to a plurality of storage containers via a controllable valve system, the containers containing a test fluid or a rinsing fluid.
This measure has the advantage that with respect to the perfusion apparatus fully automatic measurements may be conducted with the already mentioned advantages.
It is particularly preferred when the storage container is arranged above the perfusion inlet.
In that case a pump is, namely, not necessary because after opening a valve in a respective connecting conduit the test or rinsing liquid may flow to the perfusion inlet under the action of gravity alone.
In another group of embodiments the perfusion conduit is a perfusion outlet.
This measure has the advantage that the perfusion agent that is no more required may be disposed off in a controlled manner so that in particular a spill over in the receptacle for the cell is avoided.
For that purpose the perfusion outlet is preferably provided with a second end opening being located above the first end opening.
This measure has the advantage that the cell is continuously supplied with fresh perfusion agent via the distance between the two end openings, i.e. either with a test liquid or with a rinsing liquid between the measuring steps.
Moreover, this measure has the advantage that also outside the measuring operations as such the perfusion agent may be supplied in a controlled manner in order to protect those cells that are not subjected to measurements against becoming dried out. In that case a predetermined level of liquid above the cells may be set by appropriately setting the vertical distance between the two end openings.
A particularly good effect is achieved when the two openings are oriented in opposite directions.
This measure has the advantage that a short circuit between the inlet and the outlet system is avoided because the inlet and the outlet are effected in opposite directions.
Also in that case it is likewise preferred when the perfusion outlet is connected to a suction pump, particularly an adjustable pump.
A particularly good effect is achieved when, as viewed in the direction on to the first plane, the perfusion inlet is arranged in front of the first plane and the perfusion outlet is arranged behind the second plane. This measure has the advantage that an extremely compact and safe assembly is generated in which all components necessary for the measurements may cooperate in an optimal way.
In embodiments of the invention it is preferred when at least one measuring head is arranged on an actuator and the actuator is adapted to be displaced along a coordinate system above the receptacles for the cells.
This measure has the advantage that the measuring head may be displaced along all directions of the coordinate system in a fully automatic manner so that all necessary movements may be effected under program control. This holds true in particular for impaling the cells by means of the measuring electrodes, however, also for approaching the measuring head to the cells, if only a moisturizing of the cells with perfusion agent is required, as was explained above.
In a preferred modification of this embodiment the actuator may carry a plurality of measuring heads.
This measure has the advantage, in particular when so-called multi-well-plates are used, that several cells may be measured along a row or a column of the plate in parallel and simultaneously so that the cells contained in the wells of the plate may be measured as a whole only in a fraction of the time that would be required otherwise. When doing so it is of course possible to operate with different perfusion agents within different receptacles for the cells, i.e. with different test liquids or with different rinsing liquids. If different test liquids are used, this may mean that the same type of test liquid is used, however, in different concentrations, however, also test liquids of entirely different kinds may be utilized.
In that case it is proper to make all measuring heads displaceable relative to the actuator at least along a direction directed to the cell.
This measure has the advantage that the movements relative to a cell may be individually set, even if several cells are measured in parallel as discussed above.
In a particularly preferred embodiment of the inventive apparatus the measuring head is adapted to be plugged or screwed onto the actuator.
This measure has the advantage that a quick replacement of the measuring head has become possible without the need of modifying the entire experimental setup.
In another preferred embodiment of the invention means are provided for injecting cDNA and/or mRNA into the cell. This is preferably effected by arranging such means on the actuator.
These measures have the advantage that also during the step of injecting (being known per se) this step is automatized in the discussed manner and, as the case may be, may be effected for a plurality of cells one after the other in a controlled fashion.
It has already been mentioned that the invention in a highly preferred way may be utilized when the receptacles for the cells are configured as a standardized multi-well-plate.
In that case a particularly good effect is achieved if the individual receptacles in the plate are provided with a readable code, and the actuator comprises means for reading the code. This holds true in particular when the code is a bar code and means are configured as a bar code reading head.
This measure has the advantage that the inventive apparatus may start its measurements at a certain predetermined cell in a predetermined well at the beginning of the automatized measuring process for a plurality of cells, and may then continue its way over the multi-well-plate according to the particular program. At each individual receptacle, in particular at each individual well it is possible to check, by reading the respective bar code, whether the position of the measuring head is correct.
Further advantages will become apparent from the description and the enclosed drawing.
It goes without saying that the advantages mentioned before and those that will be explained hereinafter may not only be used in the particularly given combination, but also in other combinations or alone, without leaving the scope of the present invention.